Bacterial urinary tract infections are common human and veterinary diseases. The enteric gram negative bacilli normally reside in the intestinal tract and become pathogens when found in the urinary tract; these enteric bacilli are classified in the family of Enterobacteriacae. The primary causative agents of urinary tract infections are gram negative bacilli. Typically, these include Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Proteus mirabilis, etc. Infrequently, gram positive cocci (such as, Staphylococcus aureus and Enterococcus faecalis) and other gram negative bacteria (such as, Pseudomonas aeruginosa) may be urinary pathogens. Other gram positive cocci (Staphylococcus, Streptococcus) and gram positive bacilli (diphtheriods, Bacillus subtilis) are most frequently encountered as normal urethral contaminants.
Bacteriologic testing is commonly performed on patients experiencing symptoms consistent with urinary tract infections. Microorganisms isolated from patients (human and veterinary) are tested to determine the identity of the pathogens and their susceptibility to antibiotics. Information pertaining to minimum inhibitory concentrations (MIC) or the categorical interpretations (susceptible, moderate susceptible, intermediate resistant, or resistant) of antimicrobial agents against an identified pathogen are critical for a (medical or veterinary) practitioner to confirm or select a proper treatment regime for urinary tract infections.
The clinical effectiveness of antimicrobial chemotherapy for bacterial urinary tract infections requires the correct identification of the causing pathogens and the selection of an appropriate antibiotic treatment regime to eradicate the disease-causing bacteria. The suspect pathogens are isolated by inoculating the specimen onto a culture medium, which is then incubated at 35° C. for 24–48 hours to obtain bacterial growth. The bacterial identity and its antimicrobial susceptibility are then determined by a series of subsequent biochemical tests and standard antimicrobial susceptibility tests.
Methods for routine antimicrobial susceptibility determination of the identified pathogens include the broth dilution method and the agar diffusion assay. The broth dilution method involves the inoculation of a standardized microbiological inoculum (e.g., 1–5×105 cfu/ml) of the pure bacterial isolate in question into a growth medium (typically, a cation-adjusted Mueller Hinton broth) containing a series of predetermined concentrations of a given antibiotic whose MIC is sought to be determined. The inoculated medium is incubated for 18–24 hours and observed for visible growth. The lowest antibiotic concentration that completely inhibits visible growth of the isolated organism as detected by the unaided eye is recorded as the MIC.
The agar diffusion method involves the placement of an antibiotic containing disc or an antibiotic gradient strip on the surface of an agar medium (typically Mueller Hinton agar plate) that has been inoculated with the pure isolate of the microorganism in question. The antibiotic substance then diffuses away from the disc such that the effective concentration of antibiotic varies as a function of the radius from the disc or strip. Thus, the diameter of a resulting no growth area about the disc should be proportional to the MIC.
Procedures to obtain these antibiotic susceptibility data are often time-consuming (48–72 hours), cumbersome, require highly skilled personnel, or require expensive, automatic equipment. Patients with symptoms of a urinary tract infection (in particular, feline and canine) are often treated without regard to bacteriologic finding because of time delays and cumbersome assay procedures required by conventional culture methods. This may compromise the quality of patient care and contribute to the emerging antibiotic resistant bacteria due to the improper use of antibiotics.
Thus, there is a need for improved microbiologic tests and antibiotic susceptibility tests, related materials, and related assay devices. If the test procedures could be simplified so that no highly skilled personnel were required for performing the test, and test results were obtained in a shorter period of time, it would facilitate the ability of health care practitioners to confirm or select a proper treatment regime for urinary tract infections. Earlier receipt by health care practitioners (medical or veterinary) of accurate antimicrobial susceptibility information would result in better patient care, and prevent the emerging of antibiotic resistant bacteria due to the improper use of antibiotics.
Furthermore, the use of chromogenic or fluorogenic enzyme substrates have been widely used in a varieties of microbial diagnostic applications. Edberg (U.S. Pat. No. 4,925,789) described a medium containing a nutrient indicator which, when metabolized by target bacteria, releases a moiety which imparts a color or other detectable change to the medium. Chen and Gu (U.S. Pat. No. 5,620,865) used a fluorogenic compound, 4-methylumbelliferyl-β-D-glucopyranoside, in a micro-specific medium for detecting enterococci. Townsend and Chen (U.S. application Ser. No. 08/484,593, filed Jun. 7, 1995) described the use of fluorogenic enzyme substrates cocktail to detect bacterial contamination in food products. Koumura et al. (U.S. Pat. No. 4,591,554) describes the use of 4-methylumbelliferyl derivatives fluorogenic analysis to detect and determine the number of microorganisms based on the amount of liberated umbelliferone derivatives. Perry and Miller used an umbelliferyl-conjugated N-acetyl-β-D-galctosaminide for specific identification of a pathogenic yeast, Candida albicans, (J. Clin. Micro. (1987) 25:2424–2425).
The traditional endpoint of antimicrobial susceptibility determination involves the direct visual or instrument recognition of microbial growth in either a biological matrix, e.g., broth or agar. Urban and Jarstrand used a nitroblue tetrazolium dye to determine the susceptibility of bacteria to antibiotics (J. Antimicro. Chem. (1981) 8:363–369). The SENSITITRE™ system uses an instrument capable of automatically reading antimicrobial susceptibility microdilution trays (J. Clin. Microbiol. (1985) 22:187–191). In this procedure, microbial growth and MIC are determined by the measurement of fluorescence produced by bacterial enzyme action on fluorescence substrates. It is disclosed that fluorogenic substrates for this group of bacteria are selected from 7-(N)-(aminoacyl)-7-amido-4-methylcoumarin, 4-methylumbellifery noanate, 4-methylumbelliferyl phosphate. Badal et al. (U.S. Pat. No. 5,457,030) disclosed the use of a mixture of fluorogenic substrates consisting of leucine-7-amido-4-methylcoumarin, phenylalanine-7-amido-4-methylcoumarin, and 4-methylumbelliferyl phosphate and a predetermined amount of an antimicrobial agent to form a mixture to determine the antimicrobial susceptibility of the majority of clinically significant gram positive organisms.
All these prior art approaches involve the use of a clone of a bacterial isolate obtained from clinical specimen prior to identification and antimicrobial susceptibility tests. Colonies, i.e., clones, of bacterial cultures, when prepared from the biological specimen, are harvested after a sufficient period of growth. The harvested colony is suspended in a suitable aqueous liquid for biochemical identification and antimicrobial susceptibility test.
Although 90–95% of all urinary infections are caused by a single type of organism, contaminating normal flora are often present on the patient's skin or in the environment, and these organisms can provide an arbitrary contaminant to a urinary sample. Contaminating microflora in a urine specimen are particularly prevalent in veterinary practices relative to medical practice in humans; this is because the specimen collection in veterinary practices tends to be more difficult to control with animals. In general, feline and canine urine specimens may be obtained through a number of means including cystocentesis, catheterization, manual compression of the urinary bladder and natural micturition. Cystocentesis is least likely to introduce microscopic contaminants (including microbial contamination). If the samples are collected by manual compression of bladder or natural micturition, even with the effort of collecting “mid-steam” sample, microbial contamination in the sample is expected. Although cystocentesis is recommended, other methods are often used in veterinarian practices due to the difficulty in controlling animals. Problems with the contamination of urine specimens have, in the past, prevented accurate assessments of effective antibacterial therapies for urinary tract infections. Accordingly, devices and related methods are needed which distinguish uropathogens from contaminating organisms.